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真核生物中,蛋白磷酸酶Cdc14在细胞有丝分裂过程中发挥着重要的调控作用.已有研究表明,在导致马铃薯晚疫病的致病疫霉病原菌中,Cdc14对游动孢子囊的形成过程起关键性的调控作用.但其在导致大豆根腐病的大豆疫霉病原菌中的作用机制还不清楚.实时定量PCR分析大豆疫霉PsCdc14在不同的生活史和侵染阶段的转录水平发现,PsCdc14在游动孢子囊形成、游动孢子和休止孢3个阶段具有较高的转录水平,但是在菌丝和侵染阶段转录水平很低.由此推测,PsCdc14在疫霉无性繁殖阶段发挥着重要的调控作用,进而影响病害的传播及蔓延.双链RNA(dsRNA)介导的转录后目的基因沉默是真核生物十分保守的一种调控机制.将体外合成dsRNA和聚乙二醇(PEG)介导的大豆疫霉原生质体转化技术相结合,建立了大豆疫霉dsRNA介导的瞬时基因沉默体系,并利用此体系对大豆疫霉中PsCdc14基因进行了功能分析.结果表明,体外合成的PsCdc14dsRNA转入大豆疫霉原生质体后,能够导致内源的基因转录水平显著下降,沉默转化子的游动孢子囊产量显著降低.本研究表明,利用dsRNA介导的瞬时基因沉默能够更加方便、快捷地分析大豆疫霉的基因功能,加深人们对大豆疫霉生长发育和致病机制的理解.
In eukaryotes, the protein phosphatase Cdc14 plays an important regulatory role in cell mitosis.Recent studies have shown that Cdc14 plays a key role in the formation of zoosporium in Phytophthora infestans, a pathogen causing potato late blight However, the mechanism of action of PsCdc14 in Phytophthora sojae pathogen causing soybean root rot is not clear.P real-time PCR analysis of Phytophthora sojae PsCdc14 at different transcriptional levels of life history and infection found that PsCdc14 in Three stages of zoospore formation, zoospore and nocturnal convalescence had higher transcript levels, but lower transcriptional levels in mycelium and infection stage, suggesting that PsCdc14 plays an important role in the clonal propagation of Phytophthora capsici And then affect the spread and spread of the disease.Double-stranded RNA (dsRNA) -mediated transcriptional gene silencing is a very conserved regulatory mechanism of eukaryotes.In vitro synthesis of dsRNA and polyethylene glycol (PEG) mediated And the protoplast transformation of Phytophthora sojae, we established the dsRNA-mediated transient gene silencing system of Phytophthora sojae and used this system to perform the function of PsCdc14 gene in Phytophthora sojae The results showed that transfection of PsCdc14 dsRNA into P. sojae could result in a significant decrease of endogenous gene transcription level and significantly decrease the zoospore production of the silenced transformants.This study showed that dsRNA mediated by dsRNA Transient gene silencing can more conveniently and quickly analyze the gene function of Phytophthora sojae and deepen the understanding of Phytophthora sojae growth and pathogenesis.